Vinblastine, also known as vincaleukoblastine, is an antitumor dimeric alkaloid produced by the plant Catharanthus roseus. According to the recent reports, the in vivo synthesis of vinblastine proceeds through an intermediate dimer 3', 4'-anhydrovinblastine which is generated when the monomers catharanthine and vindoline are properly coupled.
The 3', 4'-anhydrovinblastine (AVLB) which acts as precursor in the production of vinblastine according to the present process has only recently been identified and characterized. There are, however, recent publications which disclose methods for its isolation. For example, Canadian patent No. 1,094,552 issued Jan. 27, 1981 describes an extraction process by which AVLB is selectively separated using chromatography, from alkaloids precipitated en masse from ground C. roseus tissue e.g. dried leaves.
In J. Am. Chem. Soc./98:22/Oct. 27, 1976, Langlois et al describe a process in which the monomers catharanthine and vindoline are coupled to form AVLB. The coupling is performed using a peracid to generate first the N-oxide of catharanthine, followed by Polonovski-type fragmentation of the N-oxide initiated with addition of trifluoroacetic anhydride to form a dimer. A reducing agent such as sodium borohydride is then added to generate AVLB which can be isolated e.g. chromatographically.
Other methods for producing the precursor AVLB have been disclosed more recently. For example, copending U.S. patent application Ser. No. 685,704 filed Aug. 4, 1986 which issued on Oct. 18, 1988 as U.S. Pat. No. 4,778,885 discloses a process whereby catharanthine and vindoline are coupled in the presence of ferric ion. Copending U.S. patent application Ser. No. 893,018 filed Aug. 4, 1986 , now U.S. Pat. No. 4,918,011 describes AVLB production through enzymatic coupling of catharanthine and vindoline, using peroxidase in the presence of a peroxide source.
Although it has recently been suggested that AVLB is itself a useful pharmaceutical, efficient production of AVLB has been sought largely as a means for enhancing yield of vinblastine.
Processes for converting AVLB to vinblastine have been proposed in the art. A review of numerous references indicates that, while purely chemical processes can be used to generate vinblastine and assist in understanding the in vivo transformation, a perhaps more promising method, in terms of vinblastine yield, relies on biotransformation. For example, in J. Am. Chem. Soc., 100:19, Sept. 3, 1978, Scott et al confirm that blank incubation of the monomers catharanthine and vindoline results in detectable amounts of dimers such as anhydrovinblastine, vinblastine and leurosine. Dimer yield is slightly increased when the monomers are fed to 6 week old C. roseus plants. In J.C.S. Chem. Comm., 1979, Baxter et al report on the ability to transform AVLB to vinblastine using cell-free extracts of C. roseus plants. A denatured extract exhibited little biotransformation activity indicating that components in the extract were responsible for vinblastine generation. Similar findings were reported by Kutney using C. roseus tissue cultures as extract source (see Helvetica Chimica Acta, Vol. 65m Fasc. 7 p. 2088-2101 (1982)) and by McLauchlan et al Tetrahedron, Vol. 39, No. 22, pp 3777-3780, 1983 who used a different line of cultured C. roseus cells as extract source.
While the component responsible for the conversion of AVLB to vinblastine has yet to be identified specifically, Kutney et al have produced data which indicated that a C. roseus fraction having a molecular weight greater than 25,000 is likely responsible for the biotransformation of the catharanthine and vindoline monomers to vinblastine (see Helvetica Chimica Acta, Vol. 65m Fasc. 7 p. 2088-2101 (1982)). Experimental evidence provided in this paper indicates that direct incorporation of radiolabelled 3', 4'-anhydrovinblastine into vinblastine using C. roseus-derived protein fraction as catalyst was around 1.8% when incubated at pH 6.3 and room temperature. Clearly, however, greater vinblastine yields are required before processes of this type can be considered economically viable.
In is therefore an object of the present invention to provide a novel process for preparing vinblastine.
It is a further object of the present invention to provide a process for preparing vinblastine in improved yield.